Viruses depend on the host cell's translation apparatus and use diverse strategies to usurp it, to inhibit cellular translation and to evade cellular mechanisms that suppress viral translation. Some viruses disrupt the cap- binding complex eIF4F, inhibiting cap-dependent initiation, whereas eIF2 is inactivated in virus-infected host cells, impairing recruitment of initiator tRNA to the ribosome. Many viral mRNAs evade these inhibitory mechanisms by virtue of having conserved structured RNA elements that engage with components of the translation apparatus to initiate translation by non-canonical mechanisms. We will characterize three such processes - internal ribosomal entry, eIF2-independent initiation, and termination-dependent re-initiation - using in vitro reconstitution to establish factor requirements and characterize individual steps in each process. In parallel, we will use biochemical and biophysical approaches to determine the structures of novel factors, of these RNA elements, and of ribosomal initiation complexes assembled on them. Additional analysis of IRESs (internal ribosomal entry sites) will focus on the functions of conserved motifs in Type 1 (poliovirus) and Type 2 (encephalomyocarditis virus) IRESs, the mechanism of action of eIF4B and PCBP2 in initiation on them, identification of factor(s) required to expose the initiation codon sequestered at the 3'-border of the Type 1 rhinovirus IRES for ribosomal inspection, and determination of factor requirements for initiation on the structurally distinct Hepatitis A virus IRES. Analysis of eIF2-independent initiation will focus on how elements in Sindbis virus sub-genomic mRNA engage a 40S subunit so that the initiation codon is placed directly into the P- site, how Ligatin and related factors then promote binding of initiator tRNA, and whether rotavirus mRNAs use this or an unrelated mechanism to sustain translation in the absence of active eIF2. We will recapitulate termination-re-initiation on Respiratory syncytial virus M2 mRNA by in vitro reconstitution to determine which factors are required and to establish an outline mechanism for this process. Taken together, these studies will yield detailed, comprehensive insights into the interactions between viral mRNAs and components of the translation apparatus, providing a framework for understanding the mechanisms of a diversity of non-canonical initiation processes used by clinically important viruses, and for the design of inhibitors.